Saliva is the principal protective agent for the mouth and thus is of primary importance to oral health maintenance. Perturbations of salivary secretory mechanisms can consequently lead to serious oral health problems. The objective of this project is to study the membrane and cellular processes that underlie the phenomenon of salivary fluid secretion and thus to contribute to our understanding of the fluid secretory process. Because similar secretory mechanisms are thought to be common to a number of other tissues, this information should be of rather broad applicability and interest. During the present reporting period we have continued our in-depth studies of the salivary Na-K-2Cl cotransporter (NKCC1). We have also continued our work on the structural and functional properties of human presenilin 1, the putative proteolytic component of gamma-secretase. NKCC1 is the major Cl entry pathway into salivary acinar cells and thus is primarily responsible for driving Cl secretion, and thereby fluid secretion, in salivary glands. Obtaining a better understanding of the structure/function relationships of this protein and its behavior in acinar cells will greatly improve our knowledge of salivary function and dysfunction, as well as possibly provide indications of how to treat the latter. In past studies we have established that the functional unit of NKCC1 is a homodimer and that the intracellular 450 amino acid C-terminus of the protein is largely responsible for the dimerization interaction. During this reporting period we have continued our experiments aimed at identifying the dimerization motif of NKCC1. We have shown that a 20 amino acid stretch within the C-terminus is required for dimerization and have identified several specific amino acids that appear to be directly involved in this interaction. Appropriate mutations of these amino acids result in modified versions of NKCC1 that dimerize with themselves but not with wild-type NKCC1. Mutations in presenilin 1 (PS1) have been linked to cases of familial early-onset Alzheimer's disease. This protein is thought to be the proteolytic component of gamma-secretase, the protease that is responsible for the intramembrane cleavage of a number of substrates including the beta-amyloid precursor protein, the protein that is primarily responsible for the senile plaques characteristic of Alzheimer's disease. In past reporting periods we have described experiments that suggested a novel new topology for PS1 in the cell membrane. During the present reporting period we completed these studies by carrying out a final series of experiments documenting the luminal location of the extra-membrane loop between hydrophobic regions 8 and 9 of PS1. Considerable evidence indicates that PS1 is involved in intracellular calcium signaling. Specifically it has been found that the expression of Alzheimer?s disease-associated PS1 mutants results in enhanced accumulation of calcium in intracellular stores and larger agonist-induced calcium transients. It has been suggested that this effect is associated with the increased gamma-secretase activity resulting from PS1 mutations and specifically that this effect might be mediated by the intracellular fragment of APP resulting from gamma-secretase cleavage (AICD). In past studies, continued in the present reporting period, we used various inhibitors to block gamma-secretase activity in the human submandibular cell line HSG. Surprisingly, we have found no evidence for an effect of gamma-secretase inhibition on carbachol-induced calcium mobilization or store content in these cells. During the present reporting period we also carried out complementary experiments with presenilin-null cells. In these studies we found that the reconstitution of gamma-secretase activity by transfection with wild-type PS1 likewise had no significant effect on the content of the intracellular calcium stores. In a test of the specific hypothesis that the level of the intracellular fragment of APP resulting from gamma-secretase cleavage can modulate the Ca2+ content of intracellular stores, we were unable to demonstrate any effect of siRNA-mediated APP knockdown on the magnitude of carbachol-induced intracellular calcium release in HSG cells. Taken together our data cast considerable doubt on the hypothesis that there is a direct link between gamma-secretase activity and the content of intracellular Ca2+ stores.